PROJECT SUMMARY In patients with pulmonary hypertension (PH) and especially with pulmonary arterial hypertension (PAH), pulmonary artery (PA) stiffness increases right ventricular strain and ultimately leads to death from heart failure. However, despite pulmonary vasculature stiffening with increased impedance being a paramount mechano-biological regulator of PAH, no therapies currently target this mechanism. Therapy for PAH continues to focus on symptomatic treatment, vasodilatory therapy, or treatment of the underlying disease (if known). None of these addresses the fundamental changes in vascular remodeling. My long-term career goal is to become an expert in understanding the mechanisms underlying PAH, thus facilitating the identification of more effective therapies and improving outcomes. This goal directly builds on my prior research and extends my interests from the systemic to the pulmonary circulation. Our preliminary data has clearly demonstrated that Lysyl oxidase like-2 (LOXL2) is an attractive gene in pulmonary arterial stiffness and cardiac fibrosis. LOXL2, is an amine oxidase that catalyzes collagen crosslinking and contributes to matrix remodeling and stiffening. Our core hypothesis is that increased LOXL2 expression, secretion, and function contributes to the development of PA stiffening. Our objective here is to study LOXL2 biology, mechanism, and pathobiology in a hierarchical manner from cells to rodent models. We will determine how LOXL2 influences cell behavior, tissue mechanics, function, and matric generation during hypoxia. We will utilize lentiviral shRNA and CRISPR/Cas9 in rat PA smooth muscle cells. We will use magnetic torsion cytometry, cell substrate impedance sensing, and tensile testing; test collagen assembly, vasoreactivity, and compliance; and evaluate adhesion, motility, and proliferation. Furthermore, we will study the relationships between LOXL2 and the TGF-?1 pathway, as well as its role in rat models of PH and PAH, using multiple models to induce pulmonary vascular stiffening in the presence an absence of a LOXL2 inhibitor. Measurements will include PA stiffness using pressure myography and stress strain relationships in isolated vessels, right ventricular function using high-resolution echocardiography, and right ventricular function using pressure-volume loops. We will establish the therapeutic potential of LOXL2 inhibition in PAH and test its effects in models of PH and PAH. This could entail not only a new drug within an existing paradigm but a whole new approach to the treatment of these patients - altering the structural composition of the extracellular matrix. With the support of my mentors, my goal during the K08 award period is to acquire the expertise and training in a nurturing academic setting that will enable me to attain the proficiency necessary to conduct these experiments, and the independence to continue beyond the award period.